The unifying goal of this proposal is to understand in molecular detail the catalytic mechanism of the carbonic anhydrase (CA) isozymes. Investigations of the most efficient and least efficient of the human carbonic anhydrases, isozymes II and III, will determine how the properties of the active-site cavity affect the fundamental steps nf attack of zinc-bound hydroxide on CO2 and subsequent proton transfer to solution. Carefully selected residues near the zinc and in a position to influence catalysis, as well as residues unique to CA III, will be replaced by site-directed mutagenesis. The catalytic properties of each mutant will be studied in an array of kinetic methods including C02 hydration and esterase activity at steady state, exchange of 180 between C02 and water at chemical equilibrium, buffer catalysis, solvent deuterium isotope effects, and the binding of substrates, anions, and sulfonamide inhibitors. A secondary goal is to use magnetic resonance to help define properties of water in the active-site. Nuclear magnetic relaxation will be used to measure the average exchange rate and average distance from the metal of water protons exchanging between bulk solvent and the active-site cavity of Co(II)-substituted CA II and III (wild type and mutants). Carbonic anhydrase IV, a membrane-bound and major functional form of carbonic anhydrase in secretory tissues, will be cloned and the gene for this isozyme expressed, as we have done for isozymes II and III. Structure-function relationships of active site residues and their role in the catalytic mechanism of CA IV will be determined and compared with isozymes II and III. This work will lead to a basic understanding of proton transfer and the role of active-site. water including zinc-bound water in the carbonic anhydrases and by analogy in other enzymes. A better understanding of the active site, will lead to an enhanced capability in the rational design of inhibitors of CA in the control of glaucoma and hydrocephalus.